Lactoferrin is known as an iron-binding protein distributed in tear, saliva, peripheral blood, milk and the like. It has been known that lactoferrin has various physiological activities, for example, antibacterial activity against harmful bacteria, activity for promoting iron absorption at the intestine, anti-flammatory activity and so on. A review of the usefulness of lactoferrin is given in US-A-2007/161541, its contents hereby incorporated by reference.
Therefore addition of lactoferrin to foods, processed foods, medicines, cosmetics and the like or direct intake as supplement is desirable. These products generally need to be heat-treated e.g. pasteurized or sterilized, to render them microbially safe and shelf-stable for a longer time.
However, if no measures are taken, lactoferrin will show unstable to heating, and heat treatment of lactoferrin may result in denaturation of lactoferrin at physiological pH. It is reported in the art that the physiological activities of lactoferrin are almost lost by heating at 62.5° C. for 30 minutes, and complete denaturation is occurred by heating at 70° C. for 15 minutes.
As is clear from above, sufficient thermal treatment could not be applied to lactoferrin as an ingredient for utilizing its physiological activities. A liquid formulation or a stock solution of lactoferrin has to be subjected to sterilisation, in order to obtain a long shelf-life (several weeks or months before consumption).
EP 454.084 and EP 1.040.766 both teach iron-enriched lactoferrin beverages. Iron fortification is needed for persons having an anemic drift, pregnant women or nursing mothers.
Although it may not be explicitly mentioned there, the skilled person immediately recognizes the effect of iron saturation on the recovery of lactoferrin aimed for in EP'084 and EP'766. According to Brisson et al. “Effect of iron saturation on the recovery of lactoferrin in rennet whey coming from heat-treated skim milk” J. Dairy Sci. 90: 2655-2664, the lactoferrin complexes with iron, which complexation improves its thermal stability. However, the use of iron in nutritional fields is often not desirable, for instance from a taste perspective. Also, iron complexation may add to the stability of the lactoferrin, but over time denaturation may still arise. Hence, a need exists for additional and improved methods to achieve heat stabilisation.
GB 2,361,703 teaches stabilisation of vitamin B12 and/or folic acid by complexing it with lactoferrin, thus increasing the shelf life of folic acid and vitamin B12, otherwise exhibiting low heat and storage stability in the presence of oxygen. Lactoferrin is used from any source, including iron-stablized lactoferrins. However, if looking from a different perspective, i.e. searching for ways to stabilize lactoferrin, the solution to bind it to folic acid and vitamin B12 for use in foodstuffs is an undesirable one. These compounds are expensive, interfere in all kinds of metabolic pathways; related therewith, administration of these compounds to people is not unrestricted.
In order to address its heat stability, it is suggested in EP-A-437.958, in the name of Morinaga Milk Industry, to heat lactoferrin at a temperature over 60° C. at a pH between 1.0-6.5. From liquid chromatography results listed in table 1 therein, it is concluded that lactoferrin is stable when heated for 5 minutes at 60° C. under acidic conditions. The very same table also shows that the window of success dramatically narrows when increasing the temperature to 70, 80, 90 or even 100° C. Above 100° C., all lactoferrin is denaturated. All measurements are taken shortly after heat treatment. EP-A-437.958 is silent on stability of the lactoferrin over time and storage temperature The contents of EP-A-437.958 is herein incorporated by reference.
In an attempt to reproduce the results reported in EP-A-437.958, the inventors found that HPLC (high performance liquid chromatography) is in fact unsuited to determine the degree of denaturation. It was found that a better technique involves FPLC (Fast performance liquid chromatography) and/or ELISA, and that HPLC readily overestimates the degree of “undenaturation”. Hence, the results in table 1 of EP-A-437.958 are in reality not as good as claimed. Moreover, it was found that heat-stable lactoferrin in EP-A-437.958, if achieved at all, was short-lasting. Denaturation still occurred, under a prolonged storage time under certain temperatures within weeks. EP-A-437.958 itself is silent on the effect of heat treatment on shelf-life. Further, the limited success of the method advocated by Morinaga dramatically decreases when extending the heating times. This is in fact acknowledged in the recited patent itself. The observations in this paragraph are founded in the comparative examples accompanying the present invention. Finally, the present inventors observed that the method of EP-A-437.958 produces aqueous lactoferrin containing compositions that are only stable for longer time periods if kept at 7° C. or lower, thus necessitating a cooling system.
Concluding, the alleged heat-stability of lactoferrin by heat-treating it at acidic conditions in accordance with EP-A-437.958 is fairly limited, since product lines in practice often involve more severe combinations of heating time and temperature, in pasteurization. Often, use is made of boiling water, and especially in batch production heating times may easily extend. In addition, should it be required to keep the aqueous lactoferrin composition stable for longer time, e.g. months, it involves expensive and logistically difficult-to-operate cooling systems. Many applications or nutritional formulations containing lactoferrin should have a shelf-life of weeks, even months. The above method cannot fulfil this condition.
The inventors of the present invention have exerted their efforts to develop an improved method for heating lactoferrin without losing its physiological activities, and resulting in a heat-treated lactoferrin that remains stable over extended time periods, including storage temperatures above 7° C. Also, the inventors wanted to refrain from being restricted to complexing lactoferrin with iron or compounds such as vitamin B12 and folic acid.